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[目的]克隆肺炎支原体铁吸收调节蛋白(Fur)基因并纯化Fur蛋白,为研究其生物学功能奠定基础。[方法]利用Clustal Omega分析肺炎支原体Fur蛋白及其同源序列并用MEGA6.0构建进化树,通过PCR扩增Fur基因并对其进行双酶切,然后连接到p ET28a,得到重组载体p ET28a-Fur,将其转化大肠杆菌BL21(DE3),利用IPTG诱导Fur蛋白表达,并通过亲和层析纯化Fur蛋白。[结果]多序列比对和进化树分析表明Mp含有一个Fur蛋白。克隆得到大小为477 bp的Fur基因,编码159个氨基酸。得到重组表达质粒p ET28a-Fur,该质粒能在大肠杆菌中能高效表达,并纯化得到重组Fur蛋白。[结论]成功克隆获得Mp Fur基因,在大肠杆菌中高效表达并获得高纯度Fur蛋白。
[Objective] The research aimed to clone Fur gene of Mycoplasma pneumoniae and purify Fur protein, which laid the foundation for the study of its biological function. [Method] The Clustal Omega was used to analyze the Fur protein of Mycoplasma pneumoniae and its homologous sequences and the phylogenetic tree was constructed by using MEGA6.0. The Fur gene was amplified by PCR and double-digested by Fur, then ligated to p ET28a to obtain the recombinant vector p ET28a- Fur was transformed into E.coli BL21 (DE3). Fur protein was induced by IPTG and the Fur protein was purified by affinity chromatography. [Result] Multiple sequence alignment and phylogenetic tree analysis indicated that Mp contains a Fur protein. The 477 bp Fur gene was cloned and encoded 159 amino acids. The recombinant expression plasmid pET28a-Fur was obtained, which can be highly expressed in E. coli and purified to obtain recombinant Fur protein. [Conclusion] The Mp Fur gene was successfully cloned and expressed in E. coli with high purity Fur protein.